Methods And Compositions For Regenerating And Repairing Damaged Or Aged Tissue Or Organs Using Nonviable Irradiated Or Lyophilized Pluripotent Stem Cells

ABSTRACT

Compositions and methods are provided herein for regenerating and repairing damaged or aged tissue or organs using nonviable lethally irradiated or lyophilized pluripotent stem cells. In one aspect, the compositions and methods described herein provide anti-aging benefits to the skin by increasing the hydration reducing fine lines, wrinkles, and pores of the skin. Compositions and methods are also provided for promoting wound healing using lyophilized pluripotent stem cell powder. A method is provided for inducing cardiac muscle regeneration in a primate comprising delivering nonviable lethally irradiated pluripotent stem cells to damaged or aged areas of the heart. The compositions and methods include nonviable lethally irradiated or lyophilized pluripotent stem cells. In one aspect, the compositions and methods utilize nonviable pluripotent stem cells in the form of a powder, such as lyophilized stem cells.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/612,716, filed Mar. 19, 2012, which is incorporated herein byreference in its entirety.

FIELD

This disclosure relates to the repair and treatment of aged or damagedorgans and tissues.

BACKGROUND

Cells derived from pluripotent stem cells, also referred to herein asembryonic stem cells (“ESC”), are being investigated as agents to repairor rejuvenate tissue. However when injected in vivo, ESC, due to theirpluripotential, form teratomas, i.e. dysregulated, cancerous tumorgrowths.

To avoid generation of teratomas or disorderly growth in vivo, the ESCpresently being developed for transplantation are differentiated ex vivointo lineage committed adult stem cell phenotypes and/or fullydifferentiated cells. This process is time consuming and generallyrequires maintenance of Good Manufacturing Procedures (GMP) over anextended period of time. The differentiated cells must be purified toensure that there is no contamination from residual pluripotent ESC. Theprocess must also include a method to ensure desired cell identity andlot-to-lot equivalency.

Transplanted ESC-derived cells produced by current methods requirelifetime immune suppression to prevent immune mediated rejection ofESC-derived cells. Many immunosuppressants are also cytotoxic andinclude antimetabolites (azathioprine), alkylating agents(cyclophosphamide), and folic-acid antagonists (methotrexate or 6-MP).Other immunosuppressants include mycophenolate mofetil (CellCept® fromHoffmann-La Roche, Inc.) and cyclosporin. These drugs may cause numerousside effects including lethal infections.

Transplantation of ESC-derived cells requires the use of viable livecells that have a very limited shelf life. Maintaining cell viabilityrequires local on site production for immediate infusion and orcryopreservation (freezing) in agents such as DMSO before overnightshipping on dry ice to prevent thawing. The frozen cells must then bethawed, washed to remove cytotoxic cryo-preservatives like DMSO,sterility rechecked, and viability reconfirmed before infusion, all ofwhich increases expense and limits availability and practicalapplication.

SUMMARY

Methods and compositions are provided herein for regenerating and/orrepairing aged, degenerating, injured, diseased, or otherwise damagedorgans and tissues in a subject using nonviable pluripotent stem cellsthat have been either lethally irradiated or lyophilized. In this latteraspect, pluripotent stem cells (e.g., embryonic stem cells or “ESC”) arefrozen and then vacuum dried to a powder that has an indefinite shelflife at room temperature and may be applied locally or dissolved into asolution before injection in an amount effective to provide a desiredtherapeutic effect. Lyophilized ESC may be manufactured at one site, donot require cryopreservation, have an indefinite shelf life, do notrequire immune suppression, and may be distributed globally tohospitals, pharmacies, or other locations to be reconstituted andinjected on location similar to lyophilized proteins, antibodies, anddrugs.

As described herein, in one aspect, an anti-aging benefit is provided tothe skin by topical application of lyophilized human ESC to increase thehydration of human skin and reduce fine lines, wrinkles, pores, and skinroughness. In another aspect, a method is provided for promoting woundhealing using either lethally irradiated ESC or lyophilized ESC powder.In yet another aspect, a method is provided for intracardiac,intracoronary, and/or intravenous injection into the heart to inducecardiac muscle regeneration. Nonviable lethally irradiated ESC andlyophilized ESC, vesicles, or fragments thereof are immune independent,do not require tissue typing, and may be utilized to repair any tissueor organ.

Neither the lethally irradiated ESC nor lyophilized pluripotent stemcell powder described in the compositions and methods herein are able togrow or divide in vivo or in vitro. The lyophilized stem cells are inthe form of a powder of nonviable cells and or cellular contents thatcannot be reconstituted into viable cells. The powdered form does notinclude living cells following the lyophilization or freeze vacuumdesiccation process. Advantageously, the pluripotent stem cells killedwith either lethal doses of irradiation or converted into a powder bylyophilization retain capability to repair damaged or degeneratingorgans or tissues in a subject.

A composition and method is provided for an anti-aging benefit to humanskin. By one approach, the composition is a topical formulationcomprising lyophilized pluripotent stem cell powder in an amounteffective to provide a desired anti-aging cosmetic benefit to the skin.The method for providing an anti-aging benefit comprises administeringan anti-aging or cosmetic composition comprising lyophilized pluripotentstem cells in a cosmetically acceptable carrier to the skin of thesubject. For example, treatment with the lyophilized pluripotent stemcell powder is effective to provide the skin with a more youthfulappearance due to increased hydration of the skin and a reduction inpores, skin roughness, and fine lines and/or wrinkles.

In yet another aspect, a composition is provided comprising lyophilizedpluripotent stem cell powder which can be applied to a wound to promotewound healing. A method is also provided for promoting wound healingcomprising administering a therapeutically effective amount oflyophilized pluripotent stem cell powder to a wound of a subject.Promoting wound healing comprises, for example, accelerating woundclosure.

In another aspect, pluripotent stem cell powder can be used toregenerate or repair cardiac muscle that has been damaged through, forexample, age, disease, ischemia, or degeneration. In this respect, amethod is provided for regenerating cardiac muscle comprising applyinglyophilized pluripotent stem cell powder reconstituted in saline andinjected into a damaged or aged area or vessel of the heart.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating that pluripotent (embryonic) stem cellpowder as well as lethally irradiated embryonic stem cells increase therate at which murine wounds close compared to fibroblast and media onlycontrols.

FIG. 2 is a bar graph showing that ESC powder and lethally irradiatedembryonic stem cells increase the rate at which wounds close compared tofibroblast and media only controls.

FIG. 3 is a bar graph demonstrating the reduction in skin roughnessafter treatment of humans with cream containing human lyophilized ESCpowder.

FIG. 4 is a bar graph showing that treatment with cream containing humanlyophilized ESC powder increases skin moisturization in humans.

FIG. 5 is a bar graph showing that treatment with cream containing humanlyophilized ESC powder decreases skin wrinkles and fine lines in humans.

FIG. 6 is a bar graph showing that treatment with cream containing humanlyophilized ESC powder decreases pore size in humans.

FIG. 7 is a bar graph demonstrating that treatment with lyophilized ESCpowder as well as lethally irradiated ESC improves mouse cardiaccontractility and relaxation compared to control 30 days after acutemyocardial infarction. Injection of media alone (negative control) didnot improve heart function.

FIG. 8 is a bar graph showing Rhesus Macaque monkey cardiaccontractility (dp/dt+) in normal hearts and 30 days after acutemyocardial infarction and injection of either conditioned media(control) or lethally irradiated (100 Gy, single fraction) human ESC.

FIG. 9 is a bar graph showing Rhesus Macaque monkey cardiac relaxation(dp/dt−) in normal hearts and 30 days after acute myocardial infarctionand injection of either conditioned media (control) or lethallyirradiated (100 Gy, single fraction) human ESC.

FIG. 10 is a bar graph showing Rhesus Macaque monkey infarct size bymagnetic resonance imaging (MRI) 30 days after acute myocardialinfarction and injection of either conditioned media (control) orlethally irradiated (100 Gy, single fraction) human ESC.

FIG. 11 is a bar graph showing change in cardiac ejection fraction 1month after acute ischemia in monkeys treated with lethally irradiatedhuman ESC versus controls treated with conditioned media.

FIG. 12 is a bar graph showing the proliferation ability of mouse ESCtreated with different doses of radiation and different durations oftreatment.

FIG. 13 is a bar graph showing cell viability of mouse ESC followingtreatment with different doses of radiation.

FIG. 14 is a photograph of fluorescent in situ hybridization of myocytenuclei positive for ESC Y-chromosome DNA one month afterischemia-induced myocardial infarct in female mice and intra-myocardialinjection of male lethally irradiated ESCs.

FIG. 15 is an image of immunofluorescent staining for5-bromo-2-deoxyuridine (in green) one month after ischemia-inducedmyocardial infarct in female mice, with (top row) or without (bottomrow) intra-myocardial injection of male irradiated embryonic stem cells.

FIG. 16 is a heat map of 534 genes differentially expressed betweenlethally irradiated ESC treated and media treated hearts at differenttime points after acute myocardial infarction.

DETAILED DESCRIPTION

The methods and compositions provided herein are based on the discovery,as described in more detail herein, that nonviable lethally irradiatedor lyophilized pluripotent stem cells can be used to repair aged,degenerating, injured, diseased, or otherwise damaged organs and tissues(such as skin, wounds, heart, brain, spinal cord, lung, liver, andkidneys) in a subject, including primate subjects, and more particularlyin human subjects. At least for some applications, it is preferred thatthe nonviable pluripotent stem cells are in powder form (e.g.,lyophilized). It was surprisingly discovered that nonviable pluripotentstem cells or lyophilized pluripotent stem cells (which may includenonviable intact cells or fragments thereof) have therapeutic benefit.For instance, it was unexpectedly found that lyophilized pluripotentstem cells that are dead and desiccated were shown to have similareffectiveness to lethally irradiated pluripotent stem cells that areable to live for hours or at most days in the body after administrationto a subject.

The lyophilized powdered stem cells advantageously can be stored, suchas in sealed vials or ampules, for long periods of time and used on anas needed basis. Due to their indefinite shelf stability, the powderedstem cells provide great flexibility to the applications in which thelyophilized pluripotent stem cell powder can be used. The lyophilizedpluripotent stem cells in powdered form are not viable upon hydration,reconstitution, or suspension in liquid or other media beforeadministration to a subject. The powdered cells can also be administeredto a subject in powder form without reconstitution.

In one aspect, a method and composition are described for providing ananti-aging benefit to the skin. The method comprises deliveringlyophilized pluripotent stem cell powder to the skin. The anti-agingbenefit includes, for example, increased skin hydration and reduction inpores, wrinkles, fine lines, and skin roughness. The anti-aging benefitsresulting from treatment with the lyophilized pluripotent stem cellpowder provide the skin with a more youthful appearance.

In another aspect, a composition and method is provided for promotingwound healing in a subject. The wound healing composition compriseseither lethally irradiated pluripotent stem cells or lyophilized powderof pluripotent stem cells and the method comprises administering thewound healing composition to a wound of a subject. The wound may be anytype of wound, including acute or chronic internal or external injuries,for example, an abrasion, cut, puncture, incision, laceration, ulcer,burn, and the like. In one approach, the wound healing compositioncomprises nonviable pluripotent stem cells in powder form. It wassurprisingly found that nonviable pluripotent stem cells, particularlythose in powder form, are effective to accelerate closure of wounds.

Applicant's copending application, U.S. Pub. No. 2009-0214491, which isincorporated herein by reference in its entirety, includes data showingthat mitotically inactivated pluripotent stem cells have been used toimprove cardiac relaxation and contractility in ischemic murinemyocardium. The instant application includes data showing that themethods described herein are effective to repair organs and tissues inprimates using human nonviable ESC. In one aspect, a method is providedfor inducing cardiac muscle regeneration in a primate (including humans)comprising delivering nonviable pluripotent stem cells to a damaged oraged area or vessel of the heart. The methods herein have now also beenshown to be effective to repair organs and tissues using lyophilizedpluripotent stem cell powder.

Advantageously, unlike many other stem cell therapies, the beneficialeffects provided by nonviable pluripotent stem cells are not dependenton a permanent presence of the delivered stem cells in the target tissueor organ. It is presently believed that the nonviable stem cells arecleared from the body within a short period of time. The lyophilized ESCpowder comprises the dried “skeletal” ESC contents including theproteins and lipids of the pluripotent stem cells and, as such, does notinclude viable intact cells. Accordingly, the immune system of thesubject being treated with the lethally irradiated or lyophilized stemcells does not need to be suppressed during or after treatment with thestem cells. Reducing or obviating the need for life longimmunosuppression therapy can greatly improve a subject's comfort andquality of life. Advantageously, the nonviable cells do not formteratomas in vivo.

By “nonviable” is meant that the stem cells are prohibited from growingor dividing and in fact are already dead (e.g. lyophilized) or in theprocess of dying or undergoing apoptosis within a few days to weeks(e.g. lethally irradiated). As used herein, nonviable cells may beproduced by any method that causes cell death, such as lethal radiationinduced apoptosis or death from freeze drying (e.g., lyophilization). Ininstances where the stem cells are made nonviable by lyophilization orsimilar technique, the lyophilization process kills the cells and thecells are not viable for any period of time after administration to thesubject. The integrity of the nonviable stem cells may or may not bemaintained, but the cellular contents should be preserved with minimaldegradation such that function and activity of the contents issubstantially maintained.

In the methods described herein, the nonviable pluripotent stem cellsare delivered in an effective amount to a damaged or aged tissue ororgan in a subject in need of treatment. As used herein, the term“subject” includes mammals, such as but not limited to rodents, pigs,cats, dogs, and primates, and specifically includes humans. The term“effective amount” or “therapeutically effective amount” means theamount that will elicit the biological or medical response of a subjectthat is being sought by a medical doctor or other clinician. In oneaspect, the term “effective amount” is intended to mean the amount thatwill bring about a biologically meaningful improvement in the treatedtissue or organ. Data obtained from animal studies can be used informulating a range of dosages for human use. The dosage may varydepending upon the dosage form employed, sensitivity of the patient, andthe route of administration. The dosage suitable for a given subject canbe determined by one of skill in the art. Generally, dosage andadministration can be adjusted to provide or to maintain the desiredeffect. By this approach, the method includes delivering an effectiveamount to the damaged tissue or organ of a subject in need ofregeneration.

Stem Cells

Pluripotent stem cells have the potential to differentiate intoendoderm, mesoderm, and ectoderm. As used herein, “pluripotent” includespluripotent stem cells from all sources, including embryonic stem cells(ESCs), modified adult stem or somatic cells (ASCs), that is, inducedpluripotent stem cells (iPSC), and very small embryonic-like stem cells(VSELs).

Pluripotent stem cells traditionally arise from the blastocyst stage ofembryonic development and have the ability to develop into all types offetal and adult cells except perhaps for placenta. Embryonic pluripotentstem cells (ESC) generally can be isolated from a 50- to 150-cell, 4- to5-day-old post-fertilization blastocyst. While ESCs are capable ofindefinite ex vivo proliferation, they exist only transiently in vivoduring embryogenesis. Pluripotent stem cells have also been artificiallygenerated (i.e., induced pluripotent stem cells (iPSC)) from othersources, such as placenta or from genetic manipulation of adult stemcells (ASC) or even adult somatic cells. ASC are located in tissuesthroughout the body and function as a reservoir to replace damaged oraging cells. ASC are generally restricted in their differentiation tocell lineages of the organ system from which they originate (i.e.,“multipotent” stem cells), although recent research suggests that adulttissues, such as bone marrow, may harbor dormant pluripotent stem cellsreferred to as “very small embryonic-like stem cells” or “VSELs.”

Various animal ESC lines, such as, for example, NIH approved cell lineWAO9 human ESCs can be obtained commercially from WiCell ResearchInstitute, Madison, Wis. Human ESC line Cecol-14, utilized herein, canbe obtained commercially from Cecolfes, Bogota, Colombia. Of course,other embryonic stem cell lines may be used, if desired.

Adult stem cells can be isolated from mammalian tissue, including fromany adult organ, umbilical cord blood, or placenta. The adult stem cellsare multipotent, but they may be manipulated to provide pluripotent stemcells (iPSC) using conventional techniques.

ESC have great versatility but, compared to ASC, can be problematic forin vivo treatments due to the tendency of ESC to form teratoma. Incontrast, ASC normally do not form teratoma and follow traditionallineage-specific differentiation patterns, fulfilling their physiologichomologous function of replacing normal turnover, aging or damagedtissues.

In one aspect, the stem cells can be derived from mammals, such as butnot limited to rodents, pigs, cats, dogs, and primates, includinghumans.

Nonviable Pluripotent Stem Cells

The pluripotent stem cells useful herein are nonviable. Advantageously,nonviable stem cells do not form teratomas. Surprisingly, nonviablepluripotent stem cells, including pluripotent stem cells that have beenlyophilized into a powder, remain capable of repairing damaged orregenerating organs or tissues in a subject. No adverse effects havebeen noted after treatment.

Prior to inactivation, the stem cells may be grown under suitableculture conditions. By one approach, the stem cells can be plated with afeeder layer for long-term culture of the stem cells. The feeder cellsare treated so that the feeder cells do not divide during culturing.Human cells can be used as a feeder layer. For example, humanfibroblasts, such as foreskin fibroblasts, can be used as a feederlayer. In one aspect, the stem cells are grown to about 70 percentconfluence. In another aspect, the stem cells are grown on coatedplates, such as plates coated with gelatin, laminin, collagen,recombinant human proteins, such as recombinant laminin or collagen, aswell as commercially available substrates, such as MATRIGEL™ BasementMembrane Matrix from BD Biosciences, or combinations thereof, without afeeder layer for the final passages before collection for use in themethods described herein.

In one aspect, the stem cells may be made nonviable with irradiation,phototherapy, chemical treatment, and/or lyophilization. The selectionof the method of making pluripotent stem cells nonviable is notparticularly limited, but it is preferred that the method used iseffective to retain the intracellular contents of the stem cells. Forexample, the nonviable pluripotent stem cells useful herein also includenonviable fragments of pluripotent stem cells including vesicles orliposomal membrane encapsulated lyophilized pluripotent stem cell orfragments of pluripotent stem cells. These nonviable fragments may beused alone or in addition to non-viable but intact (i.e., cellularcontents remain within the cell membrane) pluripotent stem cells.

While there are a variety of techniques suitable for producing nonviablepluripotent stem cells, the following exemplary techniques are describedin more detail. Other techniques may be used, if desired.

Lyophilization.

Nonviable stem cells can be prepared by lyophilization usingconventional lyophilization techniques to provide a powdered materialthat is suitable for prolonged storage at room temperature or coldertemperatures. Generally, during lyophilization, water is removed fromthe cells after the cells are frozen and placed under vacuum so that icein the product changes directly from solid to vapor without passingthrough the liquid phase. Other methods of freeze-drying orcyrodesiccation may also be used, if desired.

By one approach, the pluripotent stem cells are prepared as cultured asdescribed above and washed, such as with PBS, before collection andlyophilization. In this respect, the lyophilized cells do not includethe media in which the cells were cultured, thereby eliminating thepossibility that the therapeutic benefit provided by the lyophilizedstem cells can be attributed to conditioned media and any secretedfactors, metabolites, proteins, or other components in the media.

An exemplary lyophilization technique that may be used is as follows.The cells are pretreated by dispersing them in a freezing solution andthen freezing in liquid nitrogen. In one aspect, trehalose may be usedas a lyoprotectant or freezing solution during the freezing step, butother lyoprotectants may also be used, if desired. The cells are thentransferred to a lyophilizer that maintains a temperature low enough tokeep the cells frozen. Vacuum is applied to lower atmospheric pressureto allow sublimation of water (i.e., transition of water from solidphase to vapor phase without forming an intermittent liquid). A varietyof lyophilizers are commercially available, from bench top manifolds tovery expensive large-scale production freeze driers. Lyophilization isadvantageous in that it provides a storage-ready and stable product ofstem cell contents in powder form with no viable residual cells ormicrobes. By one approach, the lyophilization conditions may be selectedto substantially maintain the integrity of the cellular proteins so asto preserve protein structure and function but without any viable cells.

Radiation.

In one aspect, the stem cells can be exposed to lethal doses ofradiation, e.g., 100 Gy single fraction. The precise radiation dosedelivered to the cells and length of dose is not critical so long as thecells are rendered nonviable.

Apoptotic Inducing Agent.

In yet another aspect, the stem cells can be treated with an agent thatinduces apoptosis, such as but not limited to Actinomycin D,Camptothecin, Cycloheximide, Dexamethasone, Doxorubicin, Etoposide, andcombinations thereof.

In one aspect, the methods described herein can be practiced within ashort period of time after lyophilization or lethal inactivation of thepluripotent stem cells or with stem cells that were previouslylyophilized and packaged for storage. In one aspect, the lyophilizedpluripotent stem cell powder may be dissolved or dispersed in forexample saline into a syringe, an implantation device, or the like,depending upon the intended mode of delivery and use. In certainaspects, the lyophilized pluripotent stem cells may be provided in unitdosage form, such that an amount of the composition is provided insingle or multi-dose dose containers. Advantageously, pluripotent stemcell compositions in the form of powders and lyophilisates areparticularly suited for shipping and long term storage prior to use.

The lyophilized pluripotent stem cell powder described herein may beadministered to a subject by a variety of modes of administration. Assuch, the formulation as well as the concentration of the compositionmay vary. In one aspect, the composition includes lyophilizedpluripotent stem cell powder. In another aspect, the composition mayinclude lyophilized vesicles or lysosomes from pluripotent stem cells.In one aspect, the compositions may be applied directly or topically totarget tissues or organs, or to surrounding fluid or tissue. In oneaspect, administration to the desired location may be done by catheter,infusion pump, or stent. Liquid formulations can be prepared, such as,for example, in the form of a solution or suspension in a non-toxic,parenterally-acceptable solvent or diluent. In another aspect, theformulation may be a powder or lyophilisate that is reconstituted in aliquid or other media of choice prior to use. In yet another aspect, theformulation may be in the form of an emulsion or liquid concentrate fordilution prior to administration. Exemplary pharmaceutically-acceptablecarriers include saline, phosphate buffered saline, isotonic saline,Ringer's solution, dextrose, sterile water, deionized water, glycerol,ethanol, 5% dextrose in water, and combinations thereof. Additionalroutes of delivery include topical application for treatment of the skinor wound. For topical application, the media to which the nonviable stemcells are added may include, for example, pharmaceutically acceptablecarriers, such as creams, ointments, emulsions, and aqueous solutions.

Anti-Aging Skin Treatment

Aging skin can be characterized by the onset of wrinkles and fine lines.The aging of the skin can be accelerated by environmental factors,including exposure to sunlight, diet, and smoking. In one aspect, thetopical formulation comprises an amount of pluripotent stem cellslyophilized powder effective to gradually provide a desired anti-agingor cosmetic benefit to the skin. A method is also provided for providinga desired anti-aging benefit by topical application in the form of acreme, lotion, or soap or other cosmetic preparation. Treatment withlyophilized pluripotent stem cells provides the skin with a moreyouthful and smooth appearance due to increased hydration and areduction in fine lines, wrinkles, and pore size.

A variety of types of topical formulations for imparting an anti-agingbenefit can be provided. By one approach, the topical formulation can beprovided in the form of an emulsion, such as a cream or lotion, astabilized gel, salve, ointment, dispersion, a shampoo or hairconditioner, a treatment serum, a liposomal delivery system, a topicalfacial pack or mask, a surfactant-based cleansing system, an aerosolizedor sprayed dispersion or emulsion, a skin conditioner, styling aid, or apigmented product such as makeup in liquid, cream, solid, or anhydrousform. Exemplary cosmetics include, for example, lipstick, lip balm,pressed powder, foundation, concealer, and eye creams or shadow. Byanother approach, topical formulation can be provided at a spa orcosmetic counter in the form of a moisturizer, sunscreen, anti-acneproduct, or the like. By yet another approach, the topical formulationcan be provided in a prescription or over-the-counter pharmaceuticalcomposition. Other formulations may also be provided, if desired.Appropriate cosmetically acceptable vehicles can be selected toformulate the topical composition in the desired form.

The topical formulation may comprise a variety of optional ingredients.For example, the topical formulation may include ingredients such as butnot limited to perfume, perfume solubilizing agents, preservatives,coloring agents, and additional active ingredients, if desired. In oneaspect, any additional ingredients included in the composition shouldnot negatively impact the integrity of the contents of the lyophilizedor nonviable pluripotent stem cells. In one aspect, the topicalcomposition may also include an additional active ingredient, such as,for example, skin penetrating agents, antioxidants, vitamins,provitamins, sunscreen, and derivatives thereof, including, for example,epigallocatechin gallate (EGCG), tocopheryl and ascorbyl derivatives,Vitamin B3, Vitamin B5, Vitamin C, Vitamin E, Vitamin E acetate,panthenol, retinoid, retinol, retinyl, propionate, retinyl palmitate,retinoic acid, and combinations thereof, as well as other activescommonly used in topical treatments, such as salicylic acid, andalpha-hydroxyacid. The composition may also include an exfoliatingagent, sun block, or tanning agent if desired.

The topical formulation comprises lyophilized powder of pluripotent stemcells. The optimal dose of nonviable pluripotent stem cells orpluripotent stem cell lyophilized powder may depend, at least in part,on the severity of the condition, method of delivery, and method ofinactivation of the pluripotent stem cells. By one exemplary approach,the topical formulation of lyophilized powder would be from traceamounts to milligram or greater concentrations per ounce. By oneexemplary approach, the topical formulation may include but is notlimited to about 5×10⁴ to about 1×10⁸ nonviable pluripotent stem cells,in another aspect about 5×10⁴ to about 5×10⁷ nonviable pluripotent stemcells, which may be provided in the form of nonviable intact cells orlyophilized fragments and cellular contents prepared from, for example,about 5×10⁴ to about 1×10⁸ pluripotent stem cells per ounce of thetopical formulation.

The treatment regimen for reduction of wrinkles and fine lines can varydepending on the particular needs of the subject. For example, the doseand frequency of administration of the topical formulation may depend inpart on the age of the subject, condition of the subject's skin, anddesired results after treatment. By way of non-limiting illustration,the topical formulation may be applied at least once daily. By anotherapproach, the topical formulation may be applied at least once weekly oron a less frequent basis. Some subjects may benefit from regularapplication of the formulation. For example, some subjects may benefitfrom a treatment regimen where the topical formulation is applied atleast once or twice or more daily for about 8 weeks. A shorter or longertreatment regimen may be used, if desired. Further, the topicalformulation may be applied all over the face or other portion of thebody in need of treatment, or may be targeted to specific areas in needof treatment. In some approaches, it may be advantageous to exfoliatethe skin prior to application of the topical composition. By oneexemplary approach, the hydration level of the skin can be measured by acorneometer, such as CM 825, CK (Cologne, Germany).

At least in some approaches, an effective amount of lyophilizedpluripotent stem cells is that amount which achieves statisticallysignificant improvement of skin hydration.

At least in some approaches, an effective amount of lyophilizedpluripotent stem cells is that amount which achieves statisticallysignificant decrease in pore size, fine lines, wrinkles, or skinroughness.

In another aspect, an anti-aging kit is provided. The anti-aging kit maycomprise a topical composition comprising lyophilized powder ofpluripotent stem cells in a creme, lotion, or other cosmetic formulationand an exfoliant, such as a chemical or mechanical exfoliant, including,for example, loofah, brush, sponge, facial cloth, and facial creams orwashes containing sodium bicarbonate, salicylic acid, or alpha-hydroxyacid. In another aspect, the anti-aging kit may comprise a topicalcomposition, such as a cream or lotion, inactivated pluripotent stemcells in powder form for mixing into the topical composition, andoptionally an exfoliant.

Wound Healing Treatment

In another aspect, a composition for the treatment of wounds comprisinglyophilized pluripotent stem cell powder is provided. The wound healingcomposition increases the rate of wound healing. The wound to be healedmay be a result of a variety of acute or chronic internal or externalinjuries, diseases, or ailments, including, for example, abrasions,cuts, punctures, incisions, lacerations, ulcers, burns, surgical,bullet, knife, or improvised explosive device induced wounds, and thelike.

The lyophilized pluripotent stem cell powder may be applied directly tothe wound, applied to a bandage or dressing that covers the wound, orre-suspended in solution and injected into the wound or its penumbra topromote wound healing. In one approach, the wound healing composition isadministered to a wound and then the wound can be covered with aconventional wound dressing, gauze, or bandage, if desired. In anotherapproach, the wound healing composition may be administered to the woundas a component of a bandage, transdermal patch, or bioadhesive. By oneexemplary approach, the wound healing composition can be applied to awound at least once daily until the wound has healed. By anotherapproach, the topical formulation can be applied at least once dailyafter the wound has healed to limit scarring.

By one exemplary approach, the wound healing composition includes anamount of lyophilized pluripotent stem cells effective to increase therate of wound healing. In one aspect, the wound healing compositioncomprises lyophilized pluripotent stem cells dispersed in apharmaceutically acceptable carrier. For example, the wound healingcomposition may include but is not limited to about 5×10⁴ to about 1×10⁸nonviable pluripotent stem cells, in another aspect about 5×10⁴ to about5×10⁷ nonviable pluripotent stem cells, which may be provided in theform of nonviable intact cells or lyophilized fragments and cellularcontents prepared from, for example, about 5×10⁴ to about 1×10⁸pluripotent stem cells per centimeter of tissue or ounce of the woundhealing composition.

A method for promoting wound healing in a subject is also provided. Themethod includes administering a wound healing composition comprisinglyophilized pluripotent stem cells to a wound of a subject in need ofwound healing. The method may further comprise preparing the woundhealing composition by mixing the lyophilized pluripotent stem cellswith a pharmaceutically acceptable carrier prior to administering thewound healing composition to the subject. The wound healing compositionis delivered to the wound in a therapeutically effective amount topromote wound healing. The composition for the treatment of wounds mayfurther comprise one or more additional components or agents, such asantibiotics or other antimicrobial compounds or agents and other agentsknown to improve wound healing.

Also provided herein is a kit comprising lyophilized pluripotent stemcells in powder form. The kit may include, for example, media forreconstituting, hydrating, or otherwise suspending the powderedpluripotent stem cells. The kit may also include instructions for mixingthe powdered pluripotent stem cells in the media to form a woundtreatment composition and for applying the wound treatment compositionto a wound. If desired, the kit may further comprise bandages or otherdressings.

Regeneration of Cardiac Muscle

In one aspect, lyophilized or nonviable human pluripotent stem cells canbe used to regenerate or repair cardiac muscle that has been damagedthrough age, disease, or degeneration. The affected area of the heartmay include, for example, an area of the heart impacting cardiacfunction. Short and or long term ischemia can result in myocyte death,tissue infarction, and loss of contractile function. For example, thearea to be treated may include ischemic penumbra or area bestcharacterized as hibernating myocardium. Hibernating myocardium is acondition due to acute or chronic ischemia where certain portions of themyocardium exhibit abnormal or no contractile function but the cellsremain viable.

In another aspect, lethally irradiated pluripotent stem cells orlyophilized pluripotent stem cell powder can be used, for example, incardiac muscle regeneration for a number of principal indications: (i)acute heart attacks; (ii) therapy for congestive heart failure patients;(iii) prevention of further disease for patients undergoing coronaryartery bypass graft; (iv) conductive tissue regeneration; (v) vesselsmooth muscle regeneration; (vi) valve regeneration; and (vii) to weanpatients from left ventricular assist devices implanted as a bridge totransplant and or destination therapy.

Cardiac muscle normally does not have or has only limited reparativepotential. In accordance with the compositions and methods describedherein, lethally irradiated pluripotent stem cells and lyophilizedpluripotent stem cell powder reconstituted in an agent such as salineare independently able to regenerate cardiac muscle in a subject. Inthis respect, a method is provided for regenerating or repairing cardiacmuscle comprising applying an effective amount of inactivatedpluripotent stem cells to a damaged or aged area of the heart. In oneaspect, the effective amount for cardiac muscle regeneration or repairis the amount necessary to provide or improve ejection fraction (EF),which is the ratio of stroke volume (SV) to end-diastolic volume (EDV),whereby EF=SV/EDV or decrease infarct size. In this respect, aneffective amount of lethally irradiated or lyophilized pluripotent stemcell powder may be the amount necessary to improve ejection fraction ordecrease infarct size measured by MRI or other conventional techniquesafter treatment. Generally, such improvement in ejection fraction and/ordecrease in infarct size also serve to improve the quality of life ofthe subject.

At least in some approaches, an effective amount of lyophilized ornonviable pluripotent stem cells for cardiac muscle regeneration orrepair is the amount necessary to improve cardiac ejection fraction bystatistically significant amount.

At in some approaches the amount of lyophilized or nonviable pluripotentstem cells for cardiac regeneration is the amount necessary tostatistically significantly decrease infarct size.

In one aspect, repeat injection of either lethally irradiatedpluripotent stem cells or lyophilized pluripotent stem cell powderreconstituted in a liquid, e.g., saline, are given at standard intervalsuntil therapeutic effect is optimized. The optimal dose may depend, atleast in part, on the condition of the organ or tissue and method ofdelivery. By one approach, about 5×10⁴ to about 1×10⁸ nonviablepluripotent stem cells, in another aspect about 5×10⁴ to about 5×10′nonviable pluripotent stem cells, which may be provided in the form ofnonviable intact cells or lyophilized fragments and cellular contentsprepared from, for example, about 5×10⁴ to about 1×10⁸ pluripotent stemcells, are delivered to the area of treatment in the subject. Otherdoses may also be used as needed to provide the desired therapeuticeffect. Depending on the type and extent of injury, it may be advisableto treat the damaged area as soon as possible after injury.

By one approach, treatment of the heart as described herein may providesignificant improvement in cardiac function such that no furthertreatment is necessary. By another approach, treatment of the heart mayprolong survival of the subject prior to more radical therapy, includingheart transplant.

Chaperone Effect of Pluripotent Stem Cells

Although not wishing to be limited by theory, nonviable pluripotent stemcells, vesicles or lipid encapsulated cellular compartments derivedtherefrom, or their lyophilized remnants may provide a chaperone(cell-help-cell) effect. Even though nonviable, exchange of informationsuch as intracellular or lipid based proteins, lipids, enzymes,ribonucleic acid, or new signaling pathways with recipient cells mayoccur. The chaperone effect appears to be required only transiently andappears to be provided by either lethally irradiated or lyophilizedpluripotent stem cells before being cleared from or degraded by thebody. The chaperone effect does not depend on immune acceptance of thestem cells or on the establishment of a committed or differentiated cellprogeny derived directly from the stem cells which advantageously avoidsthe need for differentiation of stem cells, which generally involvesgene therapy or cytokine/media directed ex vivo differentiation, andextensive purification of the stem cell derived somatic cells. Thelethally irradiated or lyophilized pluripotent stem cells can be useddirectly to augment organ or tissue specific regeneration withoutunwanted side effects of teratoma formation or persistence of a foreigncell. Lyophilized pluripotent stem cell powder or lethally irradiatedpluripotent stem cells may thus be viewed as a universal vehicle torepair any human or animal tissues without regard to MHC immunerestrictions or need for immune suppression.

In another aspect, nonviable or lyophilized pluripotent stem cells canprovide this cell-help-cell effect ex vivo to promote proliferation andrecovery of other cell types when grown in culture in a laboratory orcommercial facility. In this aspect, a method is provided for promotingthe ex vivo growth and expansion of human or animal cell lines, themethod including applying nonviable pluripotent stem cells as an ex vivofeeder layer to human cells grown in culture or administratinglyophilized pluripotent stem cell powder at various concentrations tothe media. The cells may be any type of human cells, including forexample endothelial cells, epithelial cells, fibroblasts, smooth musclecells, surface epithelial cells, microvascular endothelial cells,myocytes, keratinocytes, melanocytes, and the like derived from anytissue or organ. In one aspect, the nonviable pluripotent stem cells orlyophilized powder are effective to promote the proliferation and/orrecovery of the human cell line when grown in culture. The human cellline can then be delivered to a target tissue or organ in a subject inneed of treatment. The target for treatment may be a diseased or damagedtissue or organ, such as, for example, the brain, kidney, spinal cord,lung, liver, or heart.

In one aspect, nonviable or lyophilized pluripotent stem cells mayfunction as an in vivo biological matrix or scaffold to promoteendogenous repair in vivo in any tissue or organ system.

In another aspect, nonviable or lyophilized pluripotent stem cells canbe added to an artificial or ex vivo generated matrix or scaffold toimprove organ or tissue regeneration prior to transplantation of thetissue or organ.

The following examples are provided to illustrate certain aspects of thedisclosure but should not be construed as limiting the scope of thedisclosure. All publications and patents referenced herein areincorporated herein by reference in their entirety. Herein, it isdemonstrated that nonviable lethally irradiated or nonviable lyophilizedpluripotent human stem cells may provide an in vivo “feeder” or“chaperone effect.” Since this chaperone effect does not requiredifferentiation or permanent integration or persistence of pluripotentstem cells, this technology may be viewed as a universal method toinduce tissue repair, including but not limited to the heart, brain,spinal cord, lung, liver, and kidneys, without regard to MHCrestrictions or need for immune suppression.

EXAMPLES Example 1 Lyophilization of Embryonic Stem Cells

Lyophilization is a method of dehydration to preserve material so itdoes not decay and is easy to transport and store. The material is firstpretreated to concentrate it in a freeze-drying protectant, such astrehalose. The material is then frozen to very low temperatures such as−80° C., and subsequently dehydrated by sublimation under low pressurevacuum.

No living cells remain after lyophilization. The lyophilized cells canbe stored indefinitely at room temperature or colder temperatures andrehydrated, reconstituted, or otherwise suspended in media, matrix, orbandage as needed before use or used directly without rehydration inpowder form.

Example 2 Wound Healing Using Lyophilized ESC Powder

ICR (imprinting control region) mice (8 weeks old; female; body weight,20-23 g) were obtained from Harlan. The animals were randomly dividedinto four groups: 1) lyophilized nonviable murine embryonic stem cells(“ESC powder”), 2) lethally irradiated nonviable murine embryonic stemcells (“iESC”), 3) irradiated murine nonviable fibroblasts control(“fibroblast control”), and 4) murine ESC conditioned media control(“media control”). The excisional wound-splinting model was generated.There were eight animals in the media control group and nine animals ineach of the remaining groups.

After hair removal from the dorsal surface and anesthesia, two 6-mmfull-thickness excisional skin wounds were created on each side of themidline. The mice were then treated with one of the four treatments.Each mouse received the same treatment to both wounds.

Each wound treated with cells received approximately 1 million stemcells: 1.0×10⁶ cells (lethally irradiated (100 Gy) nonviable ESC orlethally irradiated (100 Gy) nonviable fibroblasts) in 60 μl of PBSinjected intradermally around the wound at four injection sites and0.3×10⁶ cells in 20 μl of growth factor-reduced Matrigel (BDBiosciences) applied onto the wound bed. The lyophilized ESC powdercontaining approximately 1 million embryonic stem cells was resuspendedin PBS and injected intradermally as described above. Conditioned mediawithout cells was also used as a control. A donut-shaped silicone splintwas placed so that the wound was centered within the splint. Animmediate-bonding adhesive (Krazy Glue, Columbus, Ohio) was used to fixthe splint to the skin, followed by interrupted sutures to stabilize itsposition, and Tegaderm (3M, London, ON, Canada) was placed over thewounds. The animals were housed individually.

Wound closure was analyzed two weeks after treatment. The wounds werephotographed and the wound opening was digitally measured. The resultsare presented in FIGS. 1 and 2. FIG. 1 includes the percent woundclosure for both wounds of each animal and FIG. 2 shows the mean percentwound closure for each group with statistically significant p value(p<0.00000001) when compared to either fibroblasts or media control. Thewounds treated with lyophilized ESC powder or lethally irradiated ESChealed very similarly and nearly 30 percent better than the fibroblastsand media control groups. It was concluded that treatment with thenonviable stem cells, either in powder or lethally irradiated form, iseffective to increase the rate at which the wounds close.

Example 3 Anti-Aging Cream Using Lyophilized Human ESC Powder

The use of face cream containing lyophilized human embryonic stem cellswas evaluated for anti-aging benefits. The face cream was prepared fromall natural components (stearic acid, emulsifying wax, oil, and water)to which the lyophilized human ESC powder was added. However, in anothermanner the lyophilized human ESC powder could be added (“mixed orstirred into”) any commercially available cream, lotion, soap, or othercosmetic product. Lyophilized embryonic stem cell powder was mixed intothe cream as a powder without hydration at a concentration of 1 millionpowder lyophilized cells per ounce of cream, although concentration ofpowder added to cream or lotion or other cosmetic base can be adjustedbased on product price range and desired effect.

The performance of the cream was evaluated over an eight week periodwith five healthy, Caucasian female test subjects between the ages of 35to 55. The test subjects abstained from using any self-tanning,anti-aging, and moisturizing products, including lotions, creams, gels,and nutritional supplements for at least 72 hours prior to thecommencement of the study and used only the experimental face creamduring the test period. The test subjects had fine lines and wrinkles ontheir faces such that any differences due to effectiveness of the creamcould be measured.

Baseline measurements were taken for each test subject prior to thefirst use of the cream. Measurements were taken thereafter at 28 and 56days of use. The test subjects were instructed to apply the creamtopically to the face twice daily. They were also instructed to use aloofah and not apply the cream to mucosal surfaces (i.e., lips, mouth,eyes) and to not apply additional makeup on top of the cream.

At the described times, surface evaluation of the skin was analyzed byVisioscan (Courage and Khazaka), which takes a direct image of theliving skin using a measuring head containing a CCD-camera and two metalhalogen lamps positioned opposite each other in order to ensure evenillumination of the measuring field on the skin. The grey leveldistribution of the pixels in the image correspond to differentphenomena (white pixels represent desquamation on the skin, dark pixelsrepresent lines and wrinkles). The software with the Visioscanautomatically calculates skin smoothness, skin roughness, scaliness andwrinkle parameters. (Fischer, T. W., et al., Direct and non-directmeasurement techniques for analysis of skin surface topography, SkinPharmacol Appl Skin Physiol 1999; 12:1-11; Grether-Beck, S., et al., AnEC-derived Tetrapeptide to Counterbalance ECM Degeneration, Cosmetic &Toiletries magazine, Vol. 124 Np. 6/June 2009, both of which areincorporated herein by reference.)

A Nova Dermal Phase Meter, Model DPM 9003 (NOVA Technology Corp.,Gloucester, Mass.) was used to obtain measurements of skin surfaceimpedance to determine electroconductivity of the treatment sites. Thismeter provides a relative measure of the retained water content of theskin as a function of the skin's dielectric value. Skin impedance wasrecorded automatically when equilibrium was achieved. (Leveque, J. L.,et al., Impedance Methods for Studying Skin Moisturization, J. Soc.Cosmet. Chem., 34: 419-428, 1983, which is incorporated herein byreference.)

Detailed, high resolution before and after digital photographs weretaken with fixed camera background, angles, settings, lighting, testsubject positioning, color bars, white balance, standardized, anddigitally certified unretouched. Photographs were evaluated using theVisioscan image analysis software which allows the evaluation parameterto be captured and quantified. The image analysis software detectedsubtle changes in color by three dimensional profile of hue, value, andchroma. These characteristics were translated into color coordinates(a*, b* and L*) whose spacing is considered with the color changesperceived by the human eye. This software also allowed wrinkles to becaptured and quantified. The size of the area of involvement differedfor each test subject; therefore, percent difference was calculatedindividually and then averaged.

None of the subjects had adverse or unexpected reactions of any kindduring treatment.

As shown in Table 1 below and in FIG. 3, treatment with the creamresulted in dramatic decreases in the Visioscan parameters of surfaceroughness (SEr) associated with the depth of fine and coarse wrinkles.The reductions were statistically significant after 28 and 56 days ofuse.

TABLE 1 Surface Evaluation of Living Skin Via Visioscan - Reduction ofFine and Coarse Wrinkles (SEr) Individual Individual % Difference %Difference Subject Baseline Day 28 Day 28 Day 56 Day 56 1 1.98 1.47−25.76% 1.35 −31.99% 2 1.40 0.95 −32.14% 0.87 −37.86% 3 2.29 1.84−19.65% 1.72 −24.89% 4 1.55 1.21 −22.10% 1.13 −27.25% 5 1.82 1.37−24.73% 1.01 −44.51% Mean 1.81 1.37 1.22 % Difference −24.36%* −32.81%*p 0.000* 0.001* t 6.767* 4.985* Max % 32.14% 44.51% Improvement*Statistically significant

As shown in Table 2 below and in FIG. 4, the Novameter readingsdemonstrated that the cream dramatically increased the skin moisturecontent. The increases were statistically significant after 28 and 56days of use.

TABLE 2 Electroconductivity Via Novameter - Skin MoisturizationEvaluation Individual Individual % Difference % Difference SubjectBaseline Day 28 Day 28 Day 56 Day 56 1 123.33 170.00 37.84% 190.0054.06% 2 158.67 197.33 24.37% 248.67 56.72% 3 200.00 286.00 43.00%288.67 44.34% 4 145.33 175.33 20.64% 182.00 25.23% 5 130.67 144.6710.71% 156.67 19.90% Mean 151.60 194.67 213.20 % Difference 28.41%*40.63%* p 0.023* 0.009* t 2.732* 3.289* Max % 43.00% 56.72% Improvement*Statistically significant

As shown in Table 3 below and FIG. 5, data obtained through the imageanalysis software demonstrated wrinkle reduction after 28 and 56 days ofusage of the cream. The results were statistically significant.

TABLE 3 Reverse Photo Engineering - Wrinkle and Fine Lines ReductionAnalysis Individual Individual % Difference % Difference SubjectBaseline Day 28 Day 28 Day 56 Day 56 1 14936 12779 −14.44% 8210 −45.03%2 53404 43101 −19.29% 34842 −34.76% 3 20991 12098 −42.37% 8181 −61.03% 425869 20736 −19.84% 17957 −30.58% 5 24916 20692 −16.95% 12659 −49.19% %Difference −22.58%* −44.12%* P 0.005* T 3.676* Max % −42.37% −61.03%Reduction *Statistically significant

As shown in FIG. 6, skin pore size was also measured over the course ofthe 56 day treatment. Detailed, high resolution before and after digitalphotographs were taken, with fixed camera background, distances, angles,settings, lighting, panelist positioning, color bars, white balance,standardized and digitally certified unretouched. Each stage in theprogression of the treatment was photographically documented and thetest area of involvement isolated. Photographs were evaluated usingimage analysis software which allows the visible pores to be capturedand quantified. As shown in FIG. 6, the mean size of the pores wasreduced from baseline by 47.9% at day 56 (p-value=0.01).

The test subjects also completed questionnaires regarding the cream aspresented in Table 4 below. The results from the questionnairecorroborated the instrumental data.

TABLE 4 Questionnaire Results Time Questions Point Agree Disagree 1)Skin appears dramatically more Day 28 60.00% 40.00% hydrated Day 5680.00% 20.00% 2) Skin feels significantly more Day 28 80.00% 20.00%hydrated Day 56 80.00% 20.00% 3) Test products significantly improve Day28 80.00% 20.00% skin's softness and smoothness Day 56 80.00% 20.00% 4)Test products significantly reduce Day 28 80.00% 20.00% roughness anddryness Day 56 80.00% 20.00% 5) Test products significantly improve Day28 80.00% 20.00% skin's radiance Day 56 80.00% 20.00% 6) Test productssignificantly improve Day 28 80.00% 20.00% skin's dullness Day 56 80.00%20.00% 7) Test products significantly improve Day 28 60.00% 40.00% skinclarity, leaving a healthy, even Day 56 60.00% 40.00% tone 8) Faceappears significantly brighter Day 28 60.00% 40.00% Day 56 80.00% 20.00%9) Test products significantly reduce Day 28 60.00% 40.00% skin'sdiscoloration Day 56 60.00% 40.00% 10) Face appears significantly moreDay 28 80.00% 20.00% youthful Day 56 60.00% 40.00% 11) Skin lookssignificantly more Day 28 80.00% 20.00% luminous Day 56 60.00% 40.00%12) Skin feels significantly firmer Day 28 60.00% 40.00% Day 56 60.00%40.00% 13) Skin looks significantly tighter Day 28 80.00% 20.00% Day 5660.00% 40.00% 14) Test products dramatically reduce Day 28 60.00% 40.00%the appearance of fine lines and Day 56 60.00% 40.00% wrinkles 15) Testproducts dramatically reduce Day 28 60.00% 40.00% the appearance ofcrow's feet around Day 56 60.00% 40.00% the eyes 16) Lines and wrinklesappear Day 28 80.00% 20.00% dramatically smoother and softer Day 5660.00% 40.00% 17) Test products significantly improve Day 28 80.00%20.00% skin's overall appearance Day 56 80.00% 20.00% 18) Test productssignificantly improve Day 28 60.00% 40.00% skin's overall health Day 5680.00% 20.00%

Example 4 Acute Myocardial Infarction Treated with Injection ofLyophilized ESC Powder

Mice underwent intraperitoneal anesthesia with a mixture of ketamine(100 mg/kg), xylazine (10 mg/kg), and atropine (0.04 mg/kg), and wereventilated via a rodent ventilator (Harvard model 687 mouse ventilator;Harvard Apparatus, Holliston, Mass.). Left thoracotomy was performed atthe fourth intercostal space. The chest wall was retracted by the use of5-0 silk or monofilament suture. Ligation proceeded with a 7-0 silksuture passed with a tapered needle underneath the left anteriordescending branch of the left coronary artery <2 mm from the tip ofnormally positioned left auricle. A 1-mm section of PE-10 tubing wasplaced on the top of the vessel, and a knot was tied on the top of thetubing to occlude the coronary artery. The knot was cut after occlusionfor 60 minutes to restore reperfusion.

The mice were divided into three groups: 1) lyophilized murine ESCpowder prepared according to Example 1 and resuspended in PBS; 2)lethally irradiated murine ESC; and 3) conditioned media control. Sixanimals were in each group.

Three intramyocardial injections of 10 μL was performed in each animalusing (depending upon group) either conditioned media, or ≈=4×10⁶lethally irradiated ESCs in PBS, or lyophilized powder from ±≈1×10⁶ ESCsrehydrated in PBS. Injections were performed into the infarction,border, and normal zones via a microsyringe. After chest wall closure,the mouse was removed from the respirator, endotracheal tube waswithdrawn, warmth was maintained by a heating pad, and 100% oxygen vianasal cone was provided under intensive care until full recovery.Cardiac contractility and relaxation were monitored via a high-fidelitytransducer-tipped pressure catheter (SPR 839; Millar Instruments,Houston, Tex.). Signals were digitized by use of a data translationseries analog digital converter and then stored and analyzed on a MillarPVAN data acquisition and analysis system. Values derived from pressuretraces were averaged over no less than 20 beats. The cardiaccontractility and relaxation data 30 days after treatment are shown asmean values of all animals in each group with P values in FIG. 7 andTable 5 below. Importantly, lyophilized ESC powder significantlyimproved heart function compared to media treated hearts and lyophilizedESC powder was as effective and not statistically different frominjection of lethally irradiated ESC in heart function. Lethallyirradiated ESC were used as a positive control because application haspreviously demonstrated that mitotically inactivated ESC are capable ofinducing endogenous murine cardiac tissue regeneration. (Burt et al:“Mitotically inactivated embryonic stem cells can be used as an in vivofeeder layer to nurse damaged myocardium after acute myocardialinfarction: a preclinical study,” Circulation Research, 2012, Oct. 26;111(10):1286-96), which is incorporated herein by reference.

TABLE 5 Cardiac Contractility and Relaxation 30 Days After AcuteMyocardial Infarction Control ESC Powder iESC Contractility 3858.337653.33 7400 (mmHg/sec) (p-value = 0.0003) Relaxation 3083.33 4603.335600 (mmHg/sec) (p-value = 0.0042)

Example 5 Cardiac Ischemia in Rhesus Macaque Monkeys Treated withLethally Irradiated ESC

Three to four year old Rhesus macaque monkeys underwent open thoractomyunder general anesthesia. After dissection and visualization, the leftanterior descending (LAD) coronary artery was ligated for approximately45 minutes. Myocardium distal to the infarct became pale, visiblyischemic, and non-contractile.

After 45 minutes, four monkeys were treated with 5×10⁶ lethallyirradiated nonviable human ESC and four monkeys were controls treatedwith conditioned media from the supernatant of human ESC by injectioninto the ischemic penumbra. The coronary artery ligation was removed andthe sternotomy closed. Cardiac function was evaluated by a high fidelitytransducer tipped catheter (Millar Instruments, Houston, Tex.) readingsand magnetic resonance imaging (MRI) of left ventricular functionperformed 30 to 45 days after infarction.

All monkeys treated with lethally irradiated ESC had rapid recoveryand/or returned to normal heart function. FIG. 8 demonstrates that 30days after acute myocardial infarction monkeys treated with conditionedmedia had a large decline in systolic contractility compared topre-infarction (maximal mean systolic contraction 4400 mmHg/sec in iESCtreated mice compared to 2300 mmHg/sec in controls). In comparison,monkeys treated with lethally irradiated human embryonic stem cells(iESC) had preservation of myocardial function one month later (mean4600 mmHg/sec to 4500 mmHg/sec) (P<0.05).

As shown in FIG. 9, the diastolic function of the heart was alsopreserved after an acute heart attack when treated with lethallyirradiated human ESC compared to media treated controls. In thecontrols, maximal mean relaxation decreased from 4300 mmHg/sec to 2600mmHg/sec. In acute ischemic hearts treated with lethally irradiatedhuman ESC maximal mean diastolic relaxation was similar beforeinfarction and 30 days after infarction (mean maximal relaxation 4800mmHg/sec versus 4600 mmHg/sec) (FIG. 9) (P<0.05).

Finally, magnetic resonance imaging (MRI) was used to determine infarctsize 30 days after acute myocardial infarction in primates treated withlethally irradiated human ESC compared to those receiving only mediaafter acute infarction (controls). FIG. 10 demonstrates virtually notissue infarct 30 days after acute ischemia in monkeys treated withlethally irradiated human ESC versus mean 22 percent infarct size inmedia controls. FIG. 11 demonstrates decreased left ventricle ejectionfraction 30 days after myocardial infarction compared to no decrease ininjection fraction in monkeys treated with lethally irradiated humanESC. MRI also demonstrated no abnormal growths or pathology in the heart8-10 months after injection of iESC.

Example 6 Irradiated ESC Fail to Proliferate or Survive Either Ex Vivoor In Vivo

Irradiation inhibited ESC proliferation and prevented ESC viability.When exposed to irradiation in culture, ESC proliferation was notdetectable after 72 hours at doses≧50 Gy (FIG. 12), and viability wasextinguished by seven days post irradiation (FIG. 13).

Example 7 iESC Exist Transiently In Vivo

To determine if injected lethally irradiated ESC survived within themyocardium of recipient mice, male (Y chromosome positive) lethallyirradiated ESC (100 Gy) were injected into the peri-infarct myocardiumof female mice. One month after coronary artery ligation, hearts werestudied for the remaining Y-chromosome by fluorescent in situhybridization (FISH). The Y chromosome was detectable in less than0.000025% (1 in 40,000) cardiac cells (FIG. 14), a level of stem cellDNA persistence previously attributable in the literature to theclinically insignificant and rare occurrence of cell fusion betweendonor stem cells and cardiomyocytes (J M Nygren et al., Bonemarrow-derived hematopoietic cells generate cardiomyocytes at a lowfrequency through cell fusion, but not transdifferentiation, Nat. Med.,10(5): 494-501 (2004)).

Example 8 In Vivo Cardiomyocyte DNA Synthesis is Increased in LethallyIrradiated ESC Treated Hearts

To determine if lethally irradiated ESC promoted cardiomyocyte DNAsynthesis in vivo, cell treated infarcted mice were injected beginningon the day of myocardial infarction once a day for 5 consecutive dayswith 5-bromo-2′-deoyuridine (BrdU). BrdU is a synthetic nucleosideanalog of thymidine that incorporates into DNA (but not RNA) during DNAsynthesis.

One hour after the surgery, mice were administered with BrdU i.p.injection daily (75 mg/kg; Sigma) for 5 days. Mice hearts were harvestedone month after surgery and fixed in 4% formaldehyde. The heart sectionswere embedded and cut into 5-um thick sections The BrdU antibody (Roche)was used to target the BrdU-positive cells in the infarcted heart,according to the manufacturer's protocol. Myocytes were identified bysarcomeric actin labeling. The percentage of BrdU positive myocytenuclei was determined by confocal fluorescent microscopy.

One month later, mice were sacrificed and myocardium analyzed for BrdU.Streaks of BrdU positive cardiomyocytes were identified within theinjured left ventricle; an average of 12 percent of cardiomyocytes werelabeled by the halogenated nucleotide in the region bordering theinfarct (FIG. 15). The corresponding value in the remote myocardium was1.7 percent. In control untreated infarcted mice, BrdU labeled myocyteswere 2 percent and 0.5 percent in the area adjacent to and distant fromthe scarred myocardium, respectively. Peri-infarct recipientcardiomyocyte DNA synthesis for iESC treated mice (12 percent) wassignificantly greater than the rare occurrence of y-chromosome iESC DNA(0.000025 percent).

Gene Expression Profile of Lethally Irradiated ESC Treated Hearts:Up-Regulation of Proliferation, Chromosome Remodeling, and MetabolicPathways; Down Regulation of Inflammatory Pathways

Total RNA was isolated from murine heart tissues by using RNeasy Minikit (Qiagen Corp., Venlo, Netherlands). Mouse hearts were placed inliquid nitrogen and grind with a motor and pestle. Tissue powder andliquid nitrogen was decanted into an Rnase-free, liquid-nitrogen-cooled,2 ml micocentrifuge tubes with Buffer RLT (Qiagen Corp.). The lysate waspipeted directly into a QIAshredder spin column (Qiagen Corp.) placed ina 2 ml collection tube, centrifuge for 2 min at full speed, andsupernatant was removed by pipetting, and transferred to a newmicrocentrifuge tube. One volume of 70% ethanol was added, mixedimmediately by pipetting, and 700 ul of sample transferred to an RNeasyspin column placed in a 2 ml collection tube and centrifuge for 15 s at8,000×g. Buffer RW1 (Qiagen Corp.) (700 ul) was added to the RNeasy spincolumn and centrifuge for 15 s at 8,000×g to wash the spin columnmembrane. The spin column membrane was washed twice by adding 500 BufferRPE (Qiagen Corp.) to the RNeasy spin column and centrifuge for 15 s at10,000×g. The RNeasy spin column was placed in a new 1.5 ml collectiontube, 30-50 ul RNase-free water was added directly to the spin columnmembrane and centrifuge for 1 min at 8,000×g to elute the RNA.

Biotin labeled cRNA was generated from high quality total RNA using theIllumina TotalPrep RNA Amplification kit (Ambion). Briefly, 150 ng oftotal RNA with 260/280 absorbance ratio was reverse transcribed with anoligo (dT) primer bearing T7 promoter. The first strand cDNA, producedin the reaction, was used to make the second strand cDNA. Purifiedsecond strand cDNA along with biotin UTPs were used to generatebiotinylated, antisense RNA of each mRNA in an in vitro transcription(IVT) reaction. Size distribution profile for the labeled cRNA sampleswere evaluated by a bioanalyzer. 750 ng of purified labeled cRNAs werehybridized at 55° C. over night with the MouseRef-8 v2 ExpressionBeadChip and washed the following day. Signals was developed withStreptavidin-Cy3 and scanned with an Illumina iScan System.

Microarray Data Analysis

RNA expression analysis was performed using the Illumina MouseRef-8BeadChip, which provides coverage of around 25,700 genes and expressedsequence tags. Heart tissue cells were treated with either media orembryonic stem cells (ESC) and their RNA was collected at various timepoints at 12 hours, 24 hours, 3 days, 7 days, and 28 days. Raw signalintensities of each probe were obtained using data analysis software(Beadstudio; Illumina) and imported to the Lumi package of Bioconductorfor data analysis before transformation and normalization (see P. Du etal., Bioinformatics, 24(13): 1547-8 (2008); S. Lin et al., Nucleic AcidsRes., 36(2): ell (2008); P. Du et al., Biol. Direct. 2: 16 (2007)). A/Pcall detection was performed based on detection p value. 13,873 out of25,697 probes with less than 0.01 were considered as valid signals. Thenthe maximum signal difference for each probe was calculated and 8167probes were kept by 2-fold cutoff. These preselected probes wereimported into time course package, EDGE (J. Leek et al., Bioinformatics,22, 507-508 (2006)), to perform a standard analysis of time dependenttreatment effects. With the threshold of 0.1 q value, 539 unique geneswere identified as significant genes involved in ESC treatment. For eachtime point, differentially expressed genes were identified using anAnalysis of Variance (ANOVA) model with empirical Bayesian varianceestimation (J. M. Wettenhall et al., Bioinformatics 20: 3705-3706(2004)). Initially, genes were identified as being differentiallyexpressed on the basis of a statistically significant (rawp-value<0.01), and 2-fold change (up or down) in expression level inESC-treated RNA samples compared to controls.

Gene expression profile from microarrays were performed to evaluatedifferences in gene expression between lethally irradiated ESC treatedhearts and conditioned media treated hearts at 12 hours and 1, 3, 7, and28 days after acute myocardial infarction (FIG. 16).

Between lethally irradiated ESC and media treated hearts, 539 genes wereexpressed differentially (>2 fold increase or decrease) at 12 hours, 39genes at 24 hours, and 7 genes at 3 days after acute myocardialinfarction. There was no difference in gene expression profile betweenconditioned media and iESC treated mice at 7 and 28 days aftertreatment. By 28 days after treatment, the gene expression profile ofiESC treated mice was no different from normal hearts.

Differences between highly expressed genes (in red) and lower expressionlevels (in green) abated over time and were not different in expressionprofile from normal (non-infarcted) hearts by 28 days after myocardialinfarction (FIG. 16). The 20 most up-regulated and down-regulated cellsignal pathways in iESC treated hearts after acute myocardial infarctionare listed below in Table 6.

TABLE 6 Most up and down regulated cell signal pathways GO (Gene Foldontogeny) IDs GO Terms P-value change Top 20 most up-regulated cellsignal pathways by iESC treatment of infarcted heart tissue GO:0044260Cellular macromolecular metabolic process 2.78e−44 163 GO:0006139Nucleobase, nucleoside, nucleotide and nucleic 7.62e−41 129 acidmetabolic process GO:0006259 DNA metabolic process  1.9e−40 53GO:0044249 Cellular biosynthetic process 1.08e−39 132 GO:0006260 DNAreplication 6.38e−38 35 GO:0022403 Cell cycle phase 1.46e−37 48GO:0000279 M phase 3.68e−35 44 GO:0000278 Mitotic cell cycle 3.16e−32 40GO:0034645 Cellular macromolecule biosynthetic process 9.16e−32 108GO:0009059 Macromolecule biosynthetic process 3.29e−31 108 GO:0007067Mitosis 1.87e−25 31 GO:0000280 Nuclear division 1.87e−25 31 GO:0000087 Mphase of mitotic cell cycle 2.52e−25 31 GO:0048285 Organelle fission3.39e−25 31 GO:0006974 Response to DNA damage stimulus 1.24e−22 32GO:0010457 Gene expression 5.94e−22 97 GO:0051276 Chromosomeorganization  1.4e−21 36 GO:0060255 Regulation of macromoleculemetabolic process 1.58e−19 80 GO:0080090 Regulation of primary metabolicprocess 3.57e−19 79 GO:0051171 Regulation of nitrogen compound metabolic6.22e−19 73 process Top 20 most down-regulated cell signal pathways byiESC treatment of infarcted heart tissue GO:0006952 Defense response13.81e−10  11 GO:0009615 Response to virus 1.31e−7  5 GO:0006954Inflammatory response 3.43e−6  6 GO:0002694 Regulation of leukocyteactivation 5.61e−6  5 GO:0050865 Regulation of cell activation 5.99−6 5GO:0008228 Opsonization 8.55e−6  2 GO:0032020 ISG-15 protein conjugation1.71e−5  2 GO:0009611 Response to wounding 3.33e−5  6 GO:0006935Chemotaxis 4.07e−5  4 GO:0001932 Regulation of protein amino acidprotein 4.67e−5  4 phosphorylation GO:0042325 Regulation ofphosphorylation 6.24e−5  5 GO:0019220 Regulation of phosphate metabolicprocess  7e−5 5 GO:0051174 Regulation of phosphorus metabolic process 7e−5 5 GO:0051249 Regulation of lymphocyte activation 9.43e−5  4GO:0050829 Defense response to gram negative bacterium .000102 2GO:0001934 Positive regulation of protein amino acid .000115 3phosphorylation GO:0031399 Regulation of protein modification process.000117 4 GO:0010562 Positive regulation of phosphorus metabolic .0001283 process GO:0042327 Positive regulation of phosphorylation .000128 3GO:0045937 Positive regulation of phosphate regulation .000128 3 process

Following acute myocardial ischemia and lethally irradiated ESCinjection, microarray cardiac expression gene profiling demonstratedup-regulation of genes involved in cell cycle, chromosome remodeling,and metabolism pathways while inflammatory pathways were down-regulated.

While the disclosure has been particularly described with reference toparticular processes and embodiments, it will be appreciated thatvarious alterations, modifications, and adaptations may be made by thoseskilled in the art based on the present disclosure and are intended tobe within the spirit and scope of the embodied method as expressed inthe appended claims.

What is claimed is:
 1. A wound healing composition comprisinglyophilized pluripotent stem cells dispersed in a pharmaceuticallyacceptable carrier and the lyophilized pluripotent stem cells includedin a therapeutically effective amount.
 2. The wound healing compositionaccording to claim 1, wherein the pluripotent stem cells are embryonicstem cells.
 3. The wound healing composition according to claim 1,wherein the lyophilized pluripotent stem cells do not includeconditioned media.
 4. The wound healing composition according to claim1, wherein the therapeutically effective amount comprises about 5×10⁴ toabout 1×10⁸ lyophilized nonviable pluripotent stem cells or cellcontents thereof.
 5. A method for promoting wound healing in a subject,the method comprising administering a wound healing compositioncomprising lyophilized pluripotent stem cells to a wound of a subject inneed of wound healing.
 6. The method according to claim 5, furthercomprising preparing the wound healing composition by mixing lyophilizedpluripotent stem cells with liquid media.
 7. The method according toclaim 5, wherein the lyophilized pluripotent stem cells do not includeconditioned media.
 8. The method according to claim 5, wherein the woundhealing composition is administered to the wound in an amount effectiveto accelerate wound closure as compared to a wound not treated with thewound healing composition.
 9. The method according to claim 5, whereinthe wound is an abrasion, cut, puncture, incision, laceration, ulcer,burn, or surgical or trauma wound.
 10. A topical anti-aging compositionfor the skin comprising lyophilized pluripotent stem cells dispersed ina pharmaceutically acceptable carrier.
 11. The topical anti-agingcomposition according to claim 10, wherein the lyophilized pluripotentstem cells do not include conditioned media.
 12. The topical anti-agingcomposition of claim 10, wherein the composition is in the form of acream, lotion, gel, salve, ointment, serum, or cosmetic.
 13. The topicalanti-aging composition of claim 10, wherein the composition furthercomprises at least one active ingredient selected from the groupconsisting of skin penetrating agents, antioxidants, vitamins,provitamins, sunscreen, and combinations thereof.
 14. The topicalanti-aging composition of claim 10, wherein the anti-aging compositioncomprises 5×10⁴ to about 1×10⁸ lyophilized pluripotent stem cells perounce of anti-aging composition.
 15. A method for providing ananti-aging effect to the skin of a subject, the method comprisingadministering an anti-aging composition comprising lyophilizedpluripotent stem cells in a cosmetically acceptable carrier to the skinof the subject.
 16. The method according to claim 15, wherein thepluripotent stem cells are embryonic stem cells.
 17. The methodaccording to claim 15, wherein the subject is a human.
 18. The methodaccording to claim 15, wherein the anti-aging composition is in a formselected from the group consisting of cream, lotion, gel, salve,ointment, serum, and cosmetic.
 19. The method according to claim 15,wherein the lyophilized pluripotent stem cells do not includeconditioned media.
 20. The method according to claim 15, wherein theanti-aging composition comprises about 5×10⁴ to about 1×10⁸ lyophilizedpluripotent stem cells per ounce of anti-aging composition.
 21. A methodfor regenerating cardiac muscle in a primate subject comprisingdelivering an effective amount of lyophilized human pluripotent stemcells or lethally irradiated human pluripotent stem cells to a damagedor aged area of the primate subject's heart.
 22. The method according toclaim 21, wherein the lyophilized pluripotent stem cells are humanembryonic stem cells.
 23. The method according to claim 21, wherein thelyophilized pluripotent stem cells are delivered by injection to thedamaged or aged area of the heart.
 24. The method according to claim 21,wherein the pluripotent stem cells are delivered in a compositioncomprising a physiologically acceptable carrier or excipient.
 25. Themethod according to claim 21, wherein the lyophilized pluripotent stemcells do not include conditioned media.
 26. A method for promoting theex vivo growth and expansion of human cell lines, the method comprisingapplying nonviable lethally irradiated or lyophilized pluripotent stemcells to human cells grown in culture.
 27. The method according to claim26, wherein the nonviable lethally irradiated or lyophilized pluripotentstem cells are applied in media or on a matrix or adherence scaffolding.28. The method according to claim 26, wherein the human cells areselected from the group consisting of endothelial cells, epithelialcells, fibroblasts, smooth muscle cells, myocytes, keratinocytes,melanocytes, and combinations thereof.